i) Field of the Invention
This invention relates to a polypeptide having anticoagulant activities like a placental coagulation inhibitor (hereinafter called "CPBII") available from human tissues led by the human placenta, a novel DNA capable of coding the polypeptide, a recombinant plasmid containing the DNA, a transformant containing the recombinant plasmid, an anticoagulant containing the polypeptide as an effective ingredient and a process for the production of the polypeptide.
ii) Description of the Background Art
Heparin, heparin cofactor-II, antithrombin-III, .alpha..sub.2 -macroglobulin, .alpha..sub.1 -trypsin inhibitor, C.sub.1 -esterase inhibitor, protein C and the like have conventionally been known as anticoagulants. It is however only heparin that has found practical utility. Heparin however has a side effect of inducing bleeding tendency. Extremely stringent limitations are therefore imposed on its manner of administration and its dosage. Heparin has hence been not satisfactory as an anticoagulant from the standpoint of safety.
Under the aforementioned circumstances, the present inventors have already succeeded in separating and purifying CPBII from the human placenta, on which an application has been filed (Japanese Patent Application Laid-open No. 96132/1988).
CPBII is a substance which has the following properties and is useful as a medicine:
(1) Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state): 73,000.+-.2,000. PA1 (2) Isoelectric point (isoelectric column electrophoresis using an ampholyte): 6.2-6.6. PA1 (3) Stability: PA1 (4) Action: PA1 (5) Analysis of amino acids:
(a) Inactivated by a heat treatment at 50.degree. C. for 30 minutes. PA2 (b) Stable in a pH range of 5.5-8.5 (37.degree. C.). PA2 (c) Stable in plasma at 37.degree. C. for 15 minutes. PA2 (a) Capable of prolonging the recalcification time. PA2 (b) Capable of prolonging the prothrombin time. PA2 (c) Capable of prolonging the activated partial thromboplastin time. PA2 The existence of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is recognized by the analysis of amino acids.
The present inventors have also prepared a monoclonal antibody specific to CPBII and already filed an application for patent thereon (Japanese Patent Application No. 86753/1988). It is feasible to perform high-sensitivity assay, purification, etc. of the CPBII by using these monoclonal antibodies.
Several problems have however arisen because human tissues typified by human placentae are presently indispensable as a raw material for obtaining CPBII. For example, there is a limitation imposed on the quantity of CPBII available form a human tissue. Difficulties are always accompanied upon collection of human tissues as a raw material, whereby stable supply of the raw material is difficult. In addition, the potential danger of pathogenic viruses which may be contained in human tissues is not ignorable.
It has hence been desired to develop a method for supplying CPBII at a lower price, in a larger volume, more stably and more safely or to develop a substance having effects similar to CPBII.